Abstract

Impromidine (IMP) and arpromidine (ARP)-derived guanidines are more potent and efficacious guinea pig (gp) histamine H2-receptor (gpH2R) than human (h) H2R agonists and histamine H1-receptor (H1R) antagonists with preference for hH1R relative to gpH1R. We examined NG-acylated imidazolylpropylguanidines (AIPGs), which are less basic than guanidines, at hH2R, gpH2R, rat H2R (rH2R), hH1R, and gpH1R expressed in Sf9 cells as probes for ligand-specific receptor conformations. AIPGs were similarly potent H2R agonists as the corresponding guanidines IMP and ARP, respectively. Exchange of pyridyl in ARP against phenyl increased AIPG potency 10-fold, yielding the most potent agonists at the hH2R-Gsα fusion protein and gpH2R-Gsα identified so far. Some AIPGs were similarly potent and efficacious at hH2R-Gsα and gpH2R-Gsα. AIPGs stabilized the ternary complex in hH2R-Gsα and gpH2R-Gsα differently than the corresponding guanidines. Guanidines, AIPGs, and small H2R agonists exhibited distinct agonist properties at hH2R, gpH2R, and rH2R measuring adenylyl cyclase activity. In contrast to ARP and IMP, AIPGs were partial H1R agonists exhibiting higher efficacies at hH1R than at gpH1R. This is remarkable because, so far, all bulky H1R agonists exhibited higher efficacies at gpH1R than at hH1R. Collectively, our data suggest that AIPGs stabilize different active conformations in hH2R, gpH2R, and rH2R than guanidines and that, in contrast to guanidines, AIPGs are capable of stabilizing a partially active state of hH1R.

H2R agonists are divided into two classes. The first class comprises small molecules related to HIS (compound 1), most importantly AMT (compound 2), and DIM (compound 3) (Fig. 1). The amino group of HIS forms an ionic interaction with Asp-98 in TM3, and the imidazole ring interacts with Tyr-182 and Asp-186 in TM5 (Gantz et al., 1992; Nederkoorn et al., 1994). Small H2R agonists activate hH2R-GsαS and gpH2R-GsαS with similar potency and efficacy (Kelley et al., 2001). The second class of H2R agonists comprises long-chained and more bulky molecules, with IMP (compound 4) and ARP (compound 6) being the prototypes (Durant et al., 1978; Buschauer, 1989). The guanidino group and the imidazolylpropyl moieties of IMP and ARP form similar interactions with H2Rs as the amino group and imidazole groups of HIS, respectively (Kelley et al., 2001). In addition, the 2-(5-methylimidazol-4-ylmethylthio)ethyl moiety of IMP and the 3-(4-fluorophenyl)-3-pyridylpropyl substituents of ARP interact with a pocket formed by multiple residues in TM3, 6, and 7 (Kelley et al., 2001). At gpH2R-GsαS, IMP and ARP are full agonists and are 30- and 16-fold more potent, respectively, than HIS. At hH2R-GsαS, IMP and ARP are only partial agonists and are just 6-fold more potent than HIS (Kelley et al., 2001). Modeling and mutagenesis studies revealed that the pharmacological differences between hH2R and gpH2R are attributable to the nonconserved Asp-271 in TM7 of gpH2R (Ala-271 in hH2Rs) and Tyr-17 in TM1 of gpH2R (Cys-17 in hH2R). Furthermore, the comparison of agonist efficacies in the GTPase assay with the efficacies of agonists at stabilizing the high-affinity ternary complex of the H2R with nucleotide-free Gsα indicated that guanidines stabilize ligand-specific H2R conformations (Kelley et al., 2001). Finally, ARP-derived compounds are H1R antagonists with a preference for gpH1R relative to hH1R, with Asn-84 in TM2 playing a crucial role in determining species selectivity of H1R ligands (Seifert et al., 2003; Bruysters et al., 2005).

Structures of H2R agonists. Compounds 1 through 3 represent small H2R agonists; compounds 4 through 17 represent bulky H2R agonists. Compounds 4 and 6 are guanidines, and compounds 5 and 7 through 17 are AIPGs. Note that compounds 4 and 5 as well as 6 and 7 represent guanidine/AIPG couples; compound 17 represents the imidazolylethyl analog of 11.

The aim of this study was to further probe the concept of ligand-specific H1R and H2R conformations. Therefore, we analyzed the interactions of H1R and H2R species isoforms with NG-acylated imidazolylpropylguanidines (AIPGs), which are less basic than guanidines (Ghorai, 2005). UR-PG146 (compound 5) is the AIPG analog of IMP (compound 4), and UR-PG136 (compound 7) is the AIPG analog of ARP (compound 6). In AIPGs 8 through 16, various substituents were introduced at the imidazolylpropyl moiety, and compound 17 represents an imidazolylethyl analog, the shorter homolog, of UR-PG80 (compound 11).

Materials and Methods

Materials. Construction of baculoviruses encoding hH2R-GsαS, gpH2R-GsαS, hH1R, and gpH1R was described previously (Kelley et al., 2001; Seifert et al., 2003). Baculoviruses encoding RGS proteins 4 and 19 were a gift from Dr. E. Ross (Department of Pharmacology, University of Southwestern Medical Center, Dallas, TX). Baculovirus encoding rH2R was a gift from Dr. C. Harteneck (Department of Pharmacology, Free University of Berlin, Berlin, Germany). Guanidines 4 and 6 were synthesized as described previously (Durant et al., 1978; Buschauer, 1989). AIPGs 5 and 7 through 17 were prepared as described previously (Ghorai, 2005). Structures of synthesized compounds were confirmed by 1H nuclear magnetic resonance spectroscopy and high-resolution mass spectrometry. Purity of compounds was >98% as determined by high-performance liquid chromatography or capillary electrophoresis (Schuster et al., 1997). AIPGs 5 and 7 through 17 were prepared as trifluoroacetate salts to ensure water solubility. Stock solutions of compounds 1 through 17 (0.1, 1, and 10 mM) each were prepared in distilled water and stored at –20°C. Under these conditions, compounds were stable for at least 2 years (longer periods of time were not studied). Further dilutions of compounds 1 through 17 were prepared fresh daily. Sources of other materials are described elsewhere (Kelley et al., 2001; Houston et al., 2002; Seifert et al., 2003). Baculovirus infection and culture of Sf9 cells and membrane preparation were performed as described previously (Kelley et al., 2001). H2R-Gsα expression levels were 5 to 6 pmol/mg as assessed by immunoblotting using the M1 monoclonal antibody and β2-adrenoceptor expressed at defined levels as standard (Kelley et al., 2001). H1R expression levels were 4 to 6 pmol/mg as assessed by [3H]mepyramine saturation binding (Seifert et al., 2003).

Miscellaneous. Protein concentrations were determined using the Bio-Rad DC protein assay kit (Bio-Rad, Hercules, CA). All analyses of experimental data were performed with Prism 4.02 software (GraphPad-Prism, San Diego, CA). Ki and KB values were calculated using the Cheng and Prusoff (1973) equation. Statistical comparisons in Table 1 were performed with the t test; statistical comparisons in Table 2 were performed with analysis of variance.

Agonist potencies and efficacies of HIS, guanidines, and AIPGs at hH2R-GsαS and gpH2R-GsαS in the GTPase assay

Steady-state GTPase activity in Sf9 membranes expressing hH2R-GsαS and gpH2R-GsαS was determined as described under Materials and Methods. Reaction mixtures contained ligands at concentrations from 1 nM to 100 μM as appropriate to generate saturated concentration-response curves. Data were analyzed by nonlinear regression and were best fit to sigmoid concentration-response curves. Typical basal GTPase activities ranged between ∼1 and 2 pmol/mg/min, and the maximal stimulatory effect of histamine (100 μM) amounted to 250 to 350% above basal. The efficacy (Emax) of histamine was determined by nonlinear regression and was set at 1.00. The Emax values of other agonists were referred to this value. Data shown are the means ± S.D. of five to eight experiments each performed in duplicate. The relative potency of histamine was set at 100, and the potencies of other agonists were referred to this value. We also calculated the ratio of the EC50 values of H2R agonists for hH2R-GsαS and gpH2R-GsαS.

Agonist potencies and efficacies of small H2R agonists, guanidines, and UR-PG61 at hH2R, gpH2R, and rH2R in the AC assay

AC activity in Sf9 membranes expressing nonfused hH2R, gpH2R, or rH2R was determined as described under Materials and Methods. Reaction mixtures contained ligands at concentrations from 1 nM to 1 mM as appropriate to generate saturated concentration/response curves. Data were analyzed by nonlinear regression and were best fit to sigmoid concentration-response curves. Typical basal and maximal HIS-stimulated AC activities were as follows: hH2R, 1.5 and 5.0 pmol/mg/min, respectively; gpH2R, 1.5 and 5.0 pmol/mg/min, respectively; and rH2R, 0.7 and 3.0 pmol/mg/min, respectively. The efficacy (Emax) of histamine was determined by nonlinear regression and was set 1.00. The Emax values of other agonists were referred to this value. Data shown are the means ± S.D. of four to five experiments performed in duplicates each.

Results

Agonist Potencies and Efficacies of Guanidines and AIPGs at hH2R-GsαS and gpH2R-GsαS in the GTPase Assay. All AIPGs studied, with the exception of compound 12 at hH2R, exhibited agonistic activity at H2R isoforms (Table 1). At hH2R-GsαS, exchange of the methylene group against a carbonyl group had little effect on the potency and efficacy of the couple 4 and 5 and moderately reduced potency but not efficacy in the couple 6 and 7. Omission of the p-fluoro substituent of the phenyl group in AIPGs (7 → 8) had little effect on potency and efficacy. The same was true for the exchange of the pyridyl group against an imidazolyl group (8 → 9). Substitution of the imidazolyl ring with an additional benzyl group (9 → 10) substantially reduced efficacy at hH2R. Most prominently, exchange of the pyridyl group against a phenyl group (8 → 11) increased potency by almost 10-fold, whereas efficacy was slightly reduced. Shortening of the linker between the carbonyl group and the phenyl rings was deleterious for agonist efficacy (11 → 12), whereas introduction of p-fluoro substituents at both phenyl rings (11 → 15, UR-PG55B) resulted in the most potent hH2R agonist known so far (25-fold more potent than HIS). Changes of the fluoro substitution pattern (compare 15 with 13 and 14) and exchange of one phenyl ring by a thiazole ring (compare 14 and 16) reduced agonist potency. Exchange of the imidazolylpropyl group against an imidazolylethyl group (11 → 17) strongly reduced efficacy at hH2R, whereas it reduced potency only moderately.

Potencies and efficacies of AIPGs were higher at gpH2R-GsαS than at hH2R-GsαS (Table 1). These differences in interaction of AIPGs with hH2R and gpH2R resulted in correlations of efficacies (Fig. 2A) and potencies (Fig. 2B) that were shifted toward gpH2R. A shift toward higher potencies and efficacies at gpH2R relative to hH2R was also observed for guanidines (Kelley et al., 2001). However, compared with the data obtained with guanidines, more AIPGs deviated from the correlation between gpH2R and hH2R. Most notably, AIPG 10 was more efficacious at hH2R than at gpH2R, and compound 17 was a similarly potent partial agonist at hH2R and gpH2R. AIPGs 10 and 15 were also just 2-fold more potent agonists at gpH2R-GsαS than at hH2R-GsαS.

Agonist Potencies and Efficacies of Small H2R Agonists, Guanidines, and UR-PG61 at hH2R, gpH2R, and rH2R in the AC Assay. Although the measurement of steady-state GTP hydrolysis at H2R-Gsα fusion proteins provides a sensitive readout for agonist potencies and efficacies independently of the effector AC, fusion proteins do not represent a physiological system (Seifert et al., 1999b). Therefore, we also determined the potencies and efficacies of representative H2R agonists at nonfused H2R isoforms by measuring AC activity (Table 2). At hH2R, DIM (compound 3) was a 5-fold less potent agonist than HIS (compound 1), whereas AMT (compound 2) was 2-fold more potent than HIS. The guanidines IMP and ARP as well as a representative AIPG (UR-PG61, compound 14) were 4- to 6-fold more potent agonists than HIS. The efficacies of compounds 2 through 4, 6, and 14 at nonfused hH2R and hH2R-GsαS were similar (Tables 1 and 2) (Kelley et al., 2001).

Correlation between efficacies and potencies of AIPGs at hH2R-GsαS and gpH2R-GsαS. Agonist efficacies were taken from Table 1, and pEC50 values were derived from the EC50 values shown in Table 1. Solid lines represent the actual correlations obtained. Dashed lines represent the 95% confidence intervals of the correlations. The straight dotted lines represent the correlations that would have been obtained if efficacies and pEC50 values, respectively, had been identical in the two systems compared with each other. The theoretical curves have a slope of 1.00. A, correlation of efficacies of AIPGs at hH2R-GsαS versus gpH2R-GsαS. Slope, 0.72 ± 0.17; r2 = 0.64; p = 0.0019 (significant). B, correlation of potencies of AIPGs at hH2R-GsαS versus gpH2R-GsαS. Slope, 1.00 ± 0.24; r2 = 0.65; p = 0.0027 (significant).

Whereas at hH2R-GsαS and gpH2R-GsαS, HIS is a similarly potent agonist (Table 1), HIS was 6-fold less potent at nonfused gpH2R than at hH2R (Table 2). A similar potency difference was observed between hH2R and rH2R. Therefore, agonist potencies among the three receptor systems could only be compared on the basis of relative agonist potencies, HIS being the reference for each H2R isoform. At gpH2R, DIM was similarly potent as HIS, whereas AMT was an almost 6-fold more potent agonist than HIS. ARP, IMP, and UR-PG61 were up to 170-fold more potent gpH2R agonists than HIS. In terms of efficacy, the compounds studied were all strong partial agonists, with DIM being the least efficacious agonist. In contrast, at gpH2R-GsαS, DIM is a full agonist (Kelley et al., 2001).

At rH2R, DIM was 2-fold less potent than HIS, whereas AMT was 6-fold more potent than HIS. IMP and ARP were up to 19-fold more potent agonists than HIS, and UR-PG61 was the most potent agonist among the compounds studied, surpassing the potency of HIS by 67-fold. At rH2R, ARP was almost a full agonist; compounds 2 through 4 were strong partial agonists, and compound 14 exhibited only moderate efficacy.

Ternary Complex Formation at hH2R-GsαS andgpH2R-GsαS. Agonists stabilize a high-affinity ternary complex with GPCR and the guanine nucleotide-free G-protein (De Lean et al., 1980; Seifert et al., 1998, 1999b). In many systems, stable GTP analogs, such as GTPγS, disrupt the ternary complex and thereby reduce the agonist affinity of GPCR (De Lean et al., 1980; Seifert et al., 1998, 1999b). Interestingly, various guanidines differentially stabilize the ternary complex at hH2R-GsαS and gpH2R-GsαS, pointing to the existence of agonist-specific H2R conformations (Kelley et al., 2001). Figure 3 shows the agonist competition curves of UR-PG146 (compound 5) and UR-PG136 (compound 7) on [3H]tiotidine (antagonist) binding to hH2R-GsαS and gpH2R-GsαS in the absence and presence of GTPγS. Table 3 shows a summary of the binding properties of compounds 5 and 7 as well as the corresponding guanidines IMP (compound 4) and ARP (compound 6). UR-PG146 did not measurably stabilize the ternary complex in hH2R-GsαS, as indicated by the missing rightward shift of the agonist competition curve in the presence of GTPγS. In contrast, GTPγS shifted the IMP competition curve at hH2R-GsαS 4-fold to the right. Compared with hH2R-GsαS, UR-PG146 was much more efficient at stabilizing the ternary complex at gpH2R-GsαS, as indicated by the high fraction of high-affinity binding sites in the absence of GTPγS and the strong rightward shift of the agonist competition curve by GTPγS. UR-PG146 stabilized the ternary complex at gpH2R-GsαS more efficiently than IMP.

Competition of [3H]tiotidine binding by AIPGs in Sf9 membranes expressing hH2R-GsαS and gpH2R-GsαS. [3H]Tiotidine binding was determined as described under Materials and Methods. Reaction mixtures contained Sf9 membranes (200–250 μg of protein per tube) expressing fusion proteins, 10 nM [3H]tiotidine, and AIPGs at the concentrations indicated on the abscissa. Reaction mixtures additionally contained distilled water (control) or GTPγS (10 μM). A and B, analysis of UR-PG136 (compound 7); C and D, analysis of UR-PG146 (compound 5). Data were analyzed for best fit to monophasic and biphasic competition curves (F test). Data points shown are the means ± S.D. of five to seven experiments performed in duplicate.

Agonist binding properties of guanidines and AIPGs at hH2R-GsαS and gpH2R-GsαS

Agonist competition binding was determined as described under Materials and Methods. Data shown in Fig. 3 were analyzed by nonlinear regression for best fit to monophasic or biphasic competition curves. Data shown are the means of five to seven experiments performed in duplicate. Numbers in parentheses represent the 95% confidence intervals. Kh and Kl designate the dissociation constants for the high- and low-affinity state of H2R, respectively. %Rh indicates the percentage of high-affinity binding sites. The corresponding values in the presence of GTPγS (10 μM) are referred to as KhGTPγS, KlGTPγS, and %RhGTPγS, respectively. If data were best fit to monophasic curves, data are listed under Kl and KlGTPγS, respectively.

In contrast to UR-PG146, UR-PG136 efficiently stabilized the ternary complex at hH2R-GsαS, as indicated by the strong rightward shift of the agonist competition curve by GTPγS. ARP also stabilized the ternary complex at hH2R-GsαS, but unlike with UR-PG136, distinct high-affinity binding sites were discriminated with ARP. UR-PG136 stabilized the ternary complex in gpH2R-GsαS less efficiently than in hH2R-GsαS, as evident from the smaller shift of the agonist competition curve by GTPγS. At gpH2R-GsαS, ARP was an efficient stabilizer of the ternary complex, but this ternary complex formation was insensitive to guanine nucleotides, as seen from the preservation of distinct high-affinity binding sites in the presence of GTPγS.

Interaction of Guanidines and AIPGs with the H1R. Because guanidines are H1R antagonists with up to 10-fold selectivity for the gpH1R relative to the hH1R (Seifert et al., 2003), we also examined the interactions of AIPGs with H1Rs. Compared with the agonist HIS, the antagonist ARP exhibited 6-fold higher affinity to hH1R in [3H]mepyramine competition binding experiments, and the affinity of ARP to gpH1R was 150-fold higher (Table 4). IMP exhibited selectivity for the gpH1R relative to the hH1R as well. Strikingly, the exchange of a methylene group against a carbonyl group (4 → 5 and 6 → 7) reduced the affinity of AIPGs for H1R up to ∼300-fold. In general, AIPGs exhibited higher affinity for gpH1R than for hH1R, but the gpH1R selectivity for AIPGs was less pronounced than for guanidines (4 → 5 and 6 → 7). In addition, in the case of UR-PG131A (compound 9) and UR-PG55B (compound 15), affinity for both H1R isoforms was similar.

Affinities of HIS, guanidines, and AIPGs at hH1R and gpH1R in the [3H]mepyramine competition binding assay

[3H]Mepyramine competition binding in Sf9 membranes expressing hH1R or gpH1R with RGS4 or RGS19 was determined as described under Materials and Methods. Reaction mixtures contained Sf9 membranes (20–25 μg of protein), 2 nM [3H]mepyramine, and unlabeled ligands at concentrations of 10 nM to 1 mM as appropriate to generate saturated competition curves. Data were analyzed by nonlinear regression and were best fit to one-site (monophasic) competition curves. Data shown are the means ± S.D. of three to five experiments performed in duplicate. The relative affinity of HIS was set to 100, and the affinities of other ligands were referred to this value. We also calculated the ratio of the KB values for hH1R and gpH1R (gp/h).

To answer the question whether AIPGs are H1R agonists or antagonists, we examined the effects of the compounds on GTPase activity (Table 5). AIPGs exhibited weak to moderate partial agonistic activity at hH1R, with UR-PG126 (compound 16) being the most efficacious compound. AIPGs were 10- to 70-fold less potent than HIS at hH1R. At gpH1R, AIPGs were considerably less efficacious partial agonists than at hH1R, rendering calculation of agonist potencies impossible. For those compounds, antagonist potencies were calculated. The GTPase antagonist studies corroborated the notion that AIPGs exhibit only low affinity for hH1R and gpH1R with KB values in the 2 to 15 μM range. Of note, in the functional antagonist assay, AIPG 11 exhibited 2-fold higher affinity for hH1R than for gpH1R.

Agonist potencies and efficacies of HIS and AIPGs and antagonist potencies of AIPGs and ARP at hH1R and gpH1R in the GTPase assay

Steady-state GTPase activity in Sf9 membranes expressing hH1R and gpH1R in the presence of the RGS proteins 4 or 19 was determined as described under Materals and Methods. Reaction mixtures contained ligands at concentrations from 1 nM to 1 mM as appropriate to generate saturated concentration-response curves. Data were analyzed by nonlinear regression and were best fit to sigmoid concentration/response curves. Typical basal GTPase activities ranged between ∼1.5 and 2.5 pmol/mg/min, and the maximal stimulatory effect of histamine (100 μM) amounted to 125 to 175% above basal. The efficacy (Emax) of histamine was determined by nonlinear regression and was set at 1.00. The Emax values of other agonists were referred to this value. Data shown are the means ± S.D. of five to eight experiments performed in duplicates each. The relative potency of histamine at hH1R was set at 100, and the potencies of other agonists were referred to this value. With several AIPGs, particularly with gpH1R, the stimulatory effects were too small to calculate agonist potencies. In those cases, efficacies with agonist at a fixed concentration (100 μM) and KB values (determined in the presence of 1 μM HIS) were calculated.

Discussion

Previous studies with HL-60 promyelocytes and H2R-Gsα fusion proteins provided the first evidence for the notion that H2R agonists stabilize distinct ligand-specific H2R conformations, i.e., multiple active H2R states (Gespach et al., 1982; Seifert et al., 1992; Kelley et al., 2001; Wenzel-Seifert et al., 2001). A multiple-state model is a fundamental concept because it implies more versatile manipulation of GPCR-mediated signaling than within a two-state model assuming a single inactive (R) and a single active (R*) state (Seifert and Wenzel-Seifert, 2002; Kenakin, 2003). An increasing number of reports indicate that ligand-specific active states are a general property of GPCRs encompassing adrenoceptors, dopamine receptors, serotonin receptors, and cannabinoid receptors (Seifert et al., 1999a, 2001; Villazon et al., 2003; Gay et al., 2004; Clarke, 2005; Mukhopadhyay and Howlett, 2005). With respect to the H1R and H2R, ARP-derived guanidines are particularly useful conformational probes because these ligands discriminate between species isoforms of those GPCRs (Kelley et al., 2001; Wenzel-Seifert et al., 2001; Seifert et al., 2003). These data prompted us to examine a series of ARP-derived compounds in which the NG-alkyl substituent was replaced against an NG-alkanoyl group (compare compounds 4 and 6 versus 5 and 7 through 17) (Fig. 1). The resulting AIPGs are less basic than the corresponding guanidines. According to a two-state model, a change in basicity would be expected to result in quantitative changes in interactions of compounds with GPCRs, whereas within the framework of a multiple-state model, qualitative changes would be expected to occur.

The AIPG UR-PG55B (compound 15) is the most potent hH2R agonist known so far, surpassing the potency of ARP, the prototypical guanidine, by almost 4-fold (Table 1). However, at gpH2R-Gsα, UR-PG80 (compound 11) rather than compound 15 is the most potent gpH2R agonist. Whereas ARP-derived guanidines exhibit similar affinity for the couples hH1R/hH2R and gpH1R/gpH2R (Kelley et al., 2001; Seifert et al., 2003), AIPGs exhibit up to 1000-fold selectivity for H2Rs relative to Hs1Rs (Tables 1, 4, and 5). These differences between AIPGs and guanidines were the first indication for distinct interactions of AIPGs with H1R and H2R.

Ionic interaction of the amino group of HIS and the guanidino group of IMP, ARP, and related compounds with Asp-98 in TM3 is important for high-affinity ligand-H2R interaction (Gantz et al., 1992; Kelley et al., 2001). Regardless of the reduced pKa values (in the range of 7–8 for AIPGs compared with ∼12.5 for guanidines), the compounds are sufficiently basic to form an ionic interaction or charge-assisted hydrogen bond of the NG-acylguanidino group with Asp-98 at physiological pH. The geometry of both series of compounds, NG-alkylguanidines and NG-acylguanidines, is sufficiently similar to assume comparable binding modes to H2Rs (Fig. 1). Surprisingly, certain AIPGs even surpass guanidines in terms of agonistic potency at H2R isoforms from various species (Tables 1 and 2). With respect to AIPG substitution, the most striking result is that the exchange of the pyridyl group against a second phenyl group (8 → 11) increased agonist potency up to 10-fold (Fig. 1; Table 1). For hH2R, the increase in affinity of AIPGs by the diphenyl substitution was expected because Ala-271 in TM7 facilitates hydrophobic interactions (Kelley et al., 2001). However, in gpH2R, the pyridyl group of ARP participates in ion dipole interactions with Asp-271, which cannot take place with a phenyl ring (Kelley et al., 2001). These data are explained by a model in which AIPG 11 adopts a different orientation in gpH2R than in guanidines, allowing it to interact with hydrophobic amino acids present in TM3, 6, and 7.

Although, in general, AIPGs are more potent and efficacious at gpH2R than at hH2R (Fig. 2), we observed some exceptions from this rule. Notably, the hH2R tolerates introduction of an additional benzyl group at the imidazolyl group better in terms of efficacy than the gpH2R (9 → 10) (Fig. 1; Table 1). In addition, the hH2R tolerates an imidazolylethyl group better concerning agonist potency than the gpH2R (11 → 17). Moreover, AIPGs 10 and 15 are similarly potent agonists at hH2R and gpH2R. The potency-enhancing effect of the second phenyl ring in both hH2R and gpH2R (8 → 11) and the distinct structure-activity relationships of AIPGs at hH2R and gpH2R (9 → 10 and 11 → 17, 10, and 15) in the GTPase assay prompted us to examine ternary complex formation because this parameter is sensitive at unmasking ligand-specific GPCR conformations (Kelley et al., 2001; Seifert et al., 2001). Indeed, although the couples IMP (compound 4)/UR-PG146 (compound 5) and ARP (compound 6)/UR-PG136 (compound 7) resemble each other with respect to efficacy in the GTPase assay (Table 1), the compounds differ substantially from each other regarding ternary complex formation (Fig. 3; Table 3). At gpH2R-GsαS, UR-PG146 was particularly efficient at ternary complex formation. Efficient ternary complex formation that is not accompanied by a correspondingly high efficacy in terms of steady-state GTP turnover is indicative of the formation of nonsignaling (frozen) ternary complexes (Seifert et al., 2001; Kenakin, 2003). The existence of ligand-specific H2R conformations stabilized by AIPGs is further supported by the fact that, at hH2R, the KlGTPγS values of UR-PG146 (compound 5) and UR-PG136 (compound 7) are similar to agonist potencies in the GTPase assay, suggesting that the low-agonist affinity state of hH2R bound to AIPG promotes efficient guanine nucleotide exchange. Efficient coupling of the low-agonist affinity state of GPCR to G-proteins in terms of guanine nucleotide exchange was also shown for the human formyl peptide receptor (Wenzel-Seifert et al., 1999). In contrast, at gpH2R, the Kh value of UR-PG146 (compound 5) resembles the EC50 value in the GTPase assay, suggesting that the high-affinity state of gpH2R bound to compound 5 mediates guanine nucleotide exchange.

We performed structure-activity relationship studies on guanidines and AIPGs with H2R-Gsα fusion proteins measuring the outcome of ligand-receptor interactions at a proximal point of the signal transduction cascade, namely the steady-state GTPase activity (Kelley et al., 2001) (Table 1). This approach ensured comparison of H2R species isoforms under identical experimental conditions, with the endogenous agonist HIS (compound 1) being similarly potent at both GPCRs. Complementary studies with nonfused hH2R, gpH2R, and rH2R in the AC assay corroborated the notion that guanidines and AIPGs are potent H2R agonists (Table 2). Surprisingly however, in contrast to fusion proteins, up to 6-fold differences in HIS potency between nonfused H2R isoforms were observed (Tables 1 and 2). In addition, at nonfused H2R, we observed species differences in potency and efficacy among the small synthetic agonists AMT (compound 2) and DIM (compound 3) (Table 2) that were not apparent in the corresponding fusion proteins (Table 2) (Kelley et al., 2001). Moreover, like the hH2R, the rH2R contains Ala-271 in TM7 (Ruat et al., 1991). Accordingly, we expected similar potencies of guanidines and AIPGs at hH2R and rH2R in the AC assay, but this was not the case using the relative potency of HIS (compound 1) as reference (Table 2). Most prominently, the relative potency of compound 14 at rH2R was more than 10-fold higher than at hH2R. Furthermore, the system with nonfused H2R did not reveal the species-differences in efficacy of guanidines and AIPGs seen in the fusion proteins (Tables 1 and 2) (Kelley et al., 2001). Instead, the small agonist DIM (compound 3) exhibited lower efficacy at gpH2R than at hH2R. The different agonist profiles of H2R in the GTPase and AC assay fit to the concept of ligand-specific GPCR conformations, which differ from each other in their ability to promote guanine nucleotide exchange at Gsα relative to AC activation. Dissociations in ligand efficacies at promoting nucleotide exchange relative to AC activation were previously reported for the β2-adrenoceptor fused to Gsα (Seifert et al., 1999a).

Whereas for the H2R, the exchange of a methylene group against a carbonyl group had little impact on ligand potency (Fig. 1; Table 1), this exchange substantially decreased the affinity of compounds for the H1R (compare couple 6/7 in Tables 4 and 5 and couple 4/5 in Table 4) and thereby increased H2R selectivity. Unexpectedly, we also observed a change in quality of the effects of AIPGs at H1R. Specifically, most AIPGs are partial hH1R agonists (Table 5), whereas guanidines are H1R antagonists (Seifert et al., 2003). The effects of AIPGs and guanidines were studied side by side and in membranes expressing hH1R and gpH1R at similar expression levels, ruling out differences in GPCR expression level or GPCR/G-protein stoichiometry accounting for the differences between the two classes of compounds. These data indicate that guanidines and AIPGs also stabilize distinct H1R conformations. It is particularly noteworthy that AIPGs exhibit higher efficacies at hH1R than at gpH1R (Table 5). Moreover, some AIPGs exhibit similar (compounds 9 and 15) or higher affinity (compound 11) for hH1R relative to gpH1R (Tables 4 and 5). All bulky agonists studied so far exhibited a preference for gpH1R relative to hH1R in terms of affinity and/or efficacy (Seifert et al., 2003).

In conclusion, AIPGs stabilize different active conformations in hH2R, gpH2R, and rH2R than guanidines. Moreover, AIPGs are more efficient at stabilizing a partially active state in hH1R than in gpH1R. Our data corroborate the concept that a multiple-state model is more appropriate to describe ligand/GPCR interactions than a two-state model.

Acknowledgments

We thank Dr. G. Georg (Department of Medicinal Chemistry, University of Kansas, KS) for continuous support and encouragement and the reviewers of this article for constructive critique.

Footnotes

This work was supported by the National Institutes of Health Center of Biomedical Research Excellence Award 1 P20 RR15563 and matching support from the State of Kansas and the University of Kansas (to R.S. and Q.-Z.Y.) and the Graduate Training Program (Graduiertenkolleg) 760 “Medicinal Chemistry: Molecular Recognition-Ligand-Receptor Interactions” of the Deutsche Forschungsgemeinschaft (to R.S. and A.B.).

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